The broad objectives of this research are to develop novel adenovirus (Ad) vectors for human gene therapy. Recombinant Ad vectors are promising agents for a number of applications including immunotherapy, cancer treatment, and gene delivery for treatment of acquired or inherited diseases. Of the more than 1,000 gene therapy clinical trials ongoing worldwide, >25% involve Ad vectors. Ad vectors may be broadly grouped into two categories: replication-competent vectors and replication-defectivevectors. Replication-competent vectors include oncolytic Ad vectors. Replication-defective Ad vectors are being used for gene-based immunotherapy, tumor suppressor gene therapy, prophylactic and therapeutic vaccine delivery, and gene therapy of acquired and inherited disease. High capacity Ad (HC-Ad) vectors are replication-defective vectors that lack all viral coding regions. HC-Ad vectors elicit a significantly reduced host immune response and have shown encouraging results in animal models for long-term transgene delivery to a variety of tissues. Large scale production of HC-Ad vectors is difficult and invariably results in contamination with helper virus. HC-Ad vectors are not in clinical trials due, in large part, to the limitations of HC-Ad vector production. Significant improvements in the production of HC-Ad vectors will be required to readily obtain clinical grade preparations. We have developed novel approaches toward optimizing the production of HC- Ad vectors. These include the use of adeno-associatedvirus (AAV) excision elements to generate an HC- Ad vector in a single step and the development of a new helper virus to reduce helper virus contamination of HC-Ad preparations. We also have utilized AAV integration elements to develop Ad vectors capable of site- specific integration. The goals of this renewal application are to significantly improve HC-Ad production using new viral vectors and helper viruses and to examine the capacity of Ad vectors to integrate site- specifically at the AAVS1 locus using AAV integrationelements.